HIV-1 genomic RNA integrity Retroviruses are the only viruses that encapsidate two copies of their genetic material. One possible reason for this is that retroviral RNA appears to be damaged prior to reverse transcription; therefore, the presence of two copies of viral genomic RNA allows for successful reverse transcription through template switching. When virion RNA is examined on non-denaturing northern blots, it runs as a single dimer band. However, when heat- denatured to disrupt the dimer linkage, virion RNA includes a monomer band of reduced intensity compared to the dimer and also a smear of more rapidly-migrating RNAs, suggesting encapsidated RNA is extensively fragmented. Our central hypothesis is that this fragmentation of HIV-1 genomic RNA is due to the action of one or more host enzymes, the absence of which would lead to virions with more intact genomic RNAs and higher infectious titers. The objective of this proposal is to identify cellular genes that are associated with HIV- 1 RNA nicking using unbiased genetic screens and to test if these represent a new class of restriction factors. The proposed work combines Telesnitsky lab expertise in retroviral RNA with Tai lab expertise in whole- genome genetic screens for cellular factors that modulate viral infection plus Kidd lab expertise in specialized library preparation and bioinformatics, and consists of two aims: In Aim 1: we will seek to identify cellular genes that are required for observed damage to encapsidated HIV-1 RNAs using a whole-genome CRISPR library encoded in a lentiviral vector, and will validate candidate genes by using genetic depletion and rescue experiments. In Aim 2: we will assess properties of virion RNA fragmentation and examine its ramifications for viral replication. If successful, this project may reveal a new class of cellular restriction factors, may help illuminate novel cellular RNA processing pathways if the identified genes have not previously been shown to participate in RNA quality control, and may provide new insight into virion RNA structures and accessibility.